The present invention may be used or licensed by the United States Government for Governmental purposes without the payment of any royalty.
The present invention relates to methods for purifying and spinning spider silks and other structural proteins. Specifically, organic acids are used to lyse recombinant cells or other biological samples (such as non-recombinantly derived cells), and significantly enrich the purity and yields of structural proteins by hydrolyzing many of the macromolecules, while leaving the structural proteins intact. In the case of silk proteins, the resulting lysate is further purified by ion-exchange or affinity chromatography and processed into an aqueous-based mixture for fiber spinning.
Spiders produce a number of silks for different functions and are therefore useful organisms to produce a variety of structural proteins. The structural fibers of the golden orb-weaver spider (Nephila clavipes), are extremely strong and flexible, and are able to absorb impact energy from flying insects without breaking. Dragline silk fibers dissipate energy over a broad area and balance stiffness, strength and extensibility. In addition, silk proteins have very low antigenicity. Therefore, silk fibers are well suited for light weight, high performance fiber, composite and medical applications. The composition of these proteins is mainly glycine, alanine, and other short side chain amino acids, which form anti-parallel beta-pleated sheets by hydrogen bonding and hydrophobic interactions; Lucas et al., Discovery 25:19 1964. Many spider silks are resistant to digestion by proteolytic enzymes; Tillinghast and Kavanaugh, Journal of Zoology 202:212 1977, and insoluble in dilute acids and bases; Mello et al., American Chemical Society Symposium Series 544, Silk Polymers: Materials, Science and Biotechnology pp 67-79, 1995. Spiders are not capable of producing sufficient quantities of proteins to enable a practical use of their potential. To solve this problem, recombinant spider silks have been expressed in E.coli; Arcidiacono et al., Applied Microbiology and Biotechnology 49:31 1998; Fahnestock and Irwin, Applied Microbiology and Biotechnology 47:23, 1997; Fahnestock and Irwin, Applied Microbiology and Biotechnology 47:33 1997; Lewis et al., Protein Expression and Purification 7:400, 1996; Prince et al., Biochemistry 34:10879 1995. However, the purification and preparation of a protein for fiber spinning has been particularly difficult due to the solubility characteristics and unique properties of spider silk and other structural proteins.
Native Nephila clavipes spider dragline fiber has been partially solubilized in hexafluoroisopropanol (HFIP) and dried to a film. A 2.5% (w/w) solution of the film in HFIP was used for spinning; Jelinski et al., Macromolecules 31:6733 1998. The spinning was conducted with a syringe pump at 6 uL/s by forcing the HFIP solution through the spinneret into a coagulation bath.
Affinity chromatography has been used for purification by binding to an engineered tag in the recombinant protein while washing away bacterial proteins; Arcidiacono et al., Applied Microbiology and Biotechnology 49:31 1998; Fahnestock and Irwin, Applied Microbiology and Biotechnology 47:23 1997; Lewis et al., Protein Expression and Purification 7:400 1996; Prince et al., Biochemistry 34:10879 1995. One commonly used tag is a hexa-histidine tag, that binds with high affinity to a nickel affinity resin. After washing away the bacterial proteins, the tagged recombinant protein can be eluted from the resin. There are several disadvantages to this method: 1) large volumes of denaturing buffers are required, involving multiple steps and time; 2) not all target protein is recovered; 3) other bacterial proteins remain, often requiring additional purification (i.e., high-performance liquid chromatography (HPLC)); 4) the method is not easily scaled-up; 5) and the presence of an affinity tag on the recombinant protein may increase its antigenicity and interfere with the necessary molecular alignment required for high strength fibers. Accordingly, there is a continuing need to develop new methods for the purification of structural proteins, spinning of silk fibers lacking the engineered tag and enabling the assembly of macromolecular structures without potential interferences.
As a solution to the above-related deficiencies in the prior art, the present invention contemplates using organic acids to purify recombinant spider silks or other non-recombinant structural proteins from E. coli bacteria while removing the unwanted bacterial proteins. The invention is based on the unique solubilization and stability characteristics of these proteins, which are resistant to acid hydrolysis for prolonged periods of time at room temperature, while many globular proteins are not. Purified protein solutions can be processed into a spinnable aqueous-based mixture for the production of fibers. The present invention also contemplates an aqueous protein spinning method that closely mimics the natural spinning process of the spider and has the potential to produce fibers with properties that may resemble or improve upon those of natural silk fibers. The present invention represents the first known example of an aqueous process for the spinning of silk proteins into fibers. Furthermore, this invention is the only known report, to date, of spinning recombinant silk proteins into fibers. The present invention displays numerous advantages over the background art, including a purification method with organic acids containing fewer steps, requiring less time and smaller volumes of reagents. The present invention also results in better recovery of protein at a higher purity. For example, the (SP1)7 protein can be recovered at a level of 150 mg/L, compared to the 7mg/L recovery rate by the current art (see Prince et al., supra). While not limited to any mechanism by which a recovery is achieved, it is believed that lower protein recovery rates by the traditional methods are caused, in part, from incomplete binding of the protein to the affinity resin. Such traditional techniques include, but are not limited to, ion exchange chromatography and affinity chromatography. The inability of these proteins to bind to the resin is most likely due to a high degree of secondary structure even in the presence of high concentrations of denaturant. Sample purity from the present invention has been obtained in the range of 94-97% as determined by amino acid analysis (see Examples 1 and 2, infra). The current art results in a wide and inconsistent range of purity ranging from 70% (Prince et al., supra), to 99% (Lewis et al., 1996, supra). While high sample purity is possible using current art by affinity chromatography, the presence of the histidine affinity tag significantly increases the antigenicity of the protein and adversely affects the properties of fibers, films, or other materials by disrupting the proper molecular orientation required within the material. Also, in many cases the current art results in samples still contaminated by other bacterial proteins, requiring additional purification such as HPLC (Prince et al.; Lewis et al., supra). Finally, the methods of the present invention are easily scaled-up, and fibers are spun in an environmentally benign solution reducing hazardous waste accumulation and cost. For example, the present invention contemplates the spinning of silk proteins in an environmentally innocuous aqueous based system. In one embodiment of the present invention, a solution of an organic acid is used to effect the lysis of bacteria and initiate purification of recombinant silks and native structural proteins. Globular proteins are hydrolyzed while the silk protein remains intact. Silk proteins remain and are concentrated into an aqueous-based mixture for fiber spinning. The embodiment may comprise the following steps: a) resuspension of the cell pellet in concentrated organic acid and dilution to 2.3N in water (+/xe2x88x92denaturant and/or surfactant) to form a homogeneous mixture; b) incubation at room temperature 1 hour with stirring and centrifugation to remove cell debris; c) reduction of volume, 10-100 fold by ultrafiltration and removal of insoluble material by centrifugation; d) dialysis and removal of insoluble material by centrifugation; e) purification by ion exchange chromatography and dialysis into processing buffer; f) concentration of solution to 11-40% (w/w) protein by ultrafiltration and spinning solution into fibers. While this embodiment is given for guidance, those of skill in the art may choose to add or delete certain steps while remaining within the spirit and scope of the present invention. For example, the purification methodology may be employed with or without the spinning of the fiber solution. Several native and recombinant structural proteins have been purified by this method. Any biological sample containing a structural protein of interest, native or recombinant, is amenable to the methodology outlined in the invention. Examples of biological samples may include, but are not limited to, E. coli cells, other bacterial cells, eukaryotic cells, a medium in which a structural protein has been secreted, bone, tissues or organs. And while many variables have been examined and optimized throughout the process, each variable and optimization exemplify variations of the overall general method. Choosing among the various parameters is highly dependent on the protein being prepared. Table 1 below lends guidance to those of skill in the art.
A variety of embodiments are contemplated. In one embodiment, the present invention contemplates a method, comprising: a) providing: i) a biological sample comprising one or more structural polypeptides; and ii) an acid; b) treating said sample with said acid under conditions such that said one or more polypeptides is recovered in a solution. A variety of structural peptides are contemplated, including but not limited to polypeptides selected from SEQ ID NO.: 2, SEQ ID NO.: 4, SEQ ID NO.: 6, SEQ ID NO.: 8, SEQ ID NO.:9, and SEQ ID NO.: 11. the peptides may be recombinant or native polypeptides.
A variety of acids are contemplated. Organic acids are preferred. In one embodiment, the present invention contemplates one or more organic acids selected from formic, acetic, propionic, butyric, and valeric acids.
It is the goal to produce fibers. Therefore, in one embodiment, the method further comprises the step of manipulating said solution under conditions such that insoluble fibers are produced. Indeed, the present invention specifically contemplates the fibers produced according to the above-described process.
The present invention specifically contemplates methods wherein recombinant structural proteins are manipulated. In one embodiment, the present invention contemplates a method, comprising: a) providing: i) host cells expressing one or more recombinant structural polypeptides, and ii) a solution comprising an organic acid; b) treating said host cells with said solution to create a mixture; c) removing insoluble material from said mixture; and d) recovering said one or more recombinant polypeptides in a solution. Again, a variety of peptides are contemplated. In one embodiment, one or more polypeptides are selected from SEQ ID NO.: 2, SEQ ID NO.: 4, SEQ ID NO.: 6, SEQ ID NO.: 8, SEQ ID NO.:9, and SEQ ID NO.: 11. Again, a variety of acids are contemplated, including but not limited to organic acids selected from formic acid, acetic acid, propionic acid, butyric acid, and valeric acid.
To produce fibers, the method involves manipulation of said recovered one or more recombinant polypeptides in said solution under conditions such that insoluble fibers are produced. The present invention specifically contemplates the fibers themselves produced according to the above-described process.
A variety of host cells are contemplated for recombinant production. Thus, in one embodiment the present invention contemplates a method, comprising: a) providing: I) bacterial cells expressing one or more recombinant structural polypeptides, and ii) a solution comprising an organic acid selected from formic acid, acetic acid, propionic acid, butyric acid, and valeric acid; b) treating said bacterial cells with said solution to create a mixture; c) removing insoluble material from said mixture; and d) recovering said one or more recombinant polypeptides in a solution. As noted above, a variety of peptides are contemplated, including but not limited to one or more polypeptides is selected from SEQ ID NO.: 2, SEQ ID NO.: 4, SEQ ID NO.: 6, SEQ ID NO.: 8, and SEQ ID NO.: 11.
To produce fibers, said recovered one or more recombinant polypeptides are manipulated under conditions such that insoluble fibers are produced. In a preferred embodiment, said manipulating comprises: a) concentrating said recovered one or more recombinant silk polypeptides to create a concentrated solution; and b) forcing said concentrated solution through a spinneret. The present invention specifically contemplated the fibers themselves which are produced according to this process.
In sum, the present invention contemplates a method, which comprises providing a biological sample composed of a polypeptide and an acid, and manipulating the biological sample under conditions such that the polypeptide is substantially purified into an aqueous-based mixture.
The method, in several embodiments, includes using polypeptides that may be selected from SEQ ID NO.: 2, SEQ ID NO.: 4, SEQ ID NO.: 6, SEQ ID NO.: 8, SEQ ID NO.: 9, and SEQ ID No.: 11 herein, although other amino acid sequences are also contemplated.
In another embodiment of the present invention, the biological sample comprises many types of polypeptides, including, but not limited to, recombinant and non-recombinant polypeptides. Structural polypeptides, such as silk polypeptides, are also contemplated.
In further embodiments of the present invention, organic acids are used to manipulate aqueous-based mixtures under conditions such that the mixtures may be processed into fibers. The organic acids that may be used include, but are not limited to, formic, acetic, propionic, butyric, and valeric acids. The present invention further contemplates the product that is achieved by the methods that are described herein.
While a variety of applications for the methods and products herein described are contemplated, the applications are not limited. For example, the compositions of the present invention may comprise any type of replacement for, or blended with, high strength light-weight synthetic polymers (e.g., kevlar(copyright)) for applications such as manufacture of skis, skateboards, and tennis rackets. The method of the present invention can also be used to create a product that can be used as a precursor to the construction of many materials, including, but not limited to, films, fibers, woven articles (e.g., clothing), sutures, ballistic protection, parachutes and parachute cords.